Discovery of Reactive Microbiota-Derived Metabolites that Inhibit Host Proteases.

The gut microbiota modulate host biology in numerous ways, but little is known about the molecular mediators of these interactions. Previously, we found a widely distributed family of nonribosomal peptide synthetase gene clusters in gut bacteria. Here, by expressing a subset of these clusters in Escherichia coli or Bacillus subtilis, we show that they encode pyrazinones and dihydropyrazinones. At least one of the 47 clusters is present in 88% of the National Institutes of Health Human Microbiome Project (NIH HMP) stool samples, and they are transcribed under conditions of host colonization. We present evidence that the active form of these molecules is the initially released peptide aldehyde, which bears potent protease inhibitory activity and selectively targets a subset of cathepsins in human cell proteomes. Our findings show that an approach combining bioinformatics, synthetic biology, and heterologous gene cluster expression can rapidly expand our knowledge of the metabolic potential of the microbiota while avoiding the challenges of cultivating fastidious commensals.

A bacterial-like Pictet-Spenglerase drives the evolution of fungi to produce ß-carboline glycosides together with separate genes.

Diverse ß-carboline (ßC) alkaloids are produced by microbes, plants, and animals with myriad bioactivities and drug potentials. However, the biosynthetic mechanism of ßCs remains largely elusive, especially regarding the hydroxyl and glucosyl modifications of ßCs. Here, we report the presence of the bacterial-like Pictet-Spenglerase gene Fcs1 in the entomopathogenic Beauveria fungi that can catalyze the biosynthesis of the ßC skeleton. The overexpression of Fcs1 in Beauveria bassiana led to the identification of six ßC methyl glycosides, termed bassicarbosides (BCSs) A-F. We verified that the cytochrome P450 (CYP) genes adjacent to Fcs1 cannot oxidize ßCs. Alternatively, the separated CYP684B2 family gene Fcs2 was identified to catalyze ßC hydroxylation together with its cofactor gene Fcs3. The functional homologue of Fcs2 is only present in the Fcs1-containing fungi and highly similar to the Fcs1-connected yet nonfunctional CYP. Both evolved quicker than those from fungi without Fcs1 homologues. Finally, the paired methyl/glucosyl transferase genes were verified to mediate the production of BCSs from hydroxy-ßCs. All these functionally verified genes are located on different chromosomes of Beauveria, which is in contrast to the typical content-clustered feature of fungal biosynthetic gene clusters (BGCs). We also found that the production of BCSs selectively contributed to fungal infection of different insect species. Our findings shed light on the biosynthetic mechanism of ßC glycosides, including the identification of a ßC hydroxylase. The results of this study also propose an evolving process of fungal BGC formation following the horizontal transfer of a bacterial gene to fungi.

Domoic acid biosynthesis in the red alga Chondria armata suggests a complex evolutionary history for toxin production.

Domoic acid (DA), the causative agent of amnesic shellfish poisoning, is produced by select organisms within two distantly related algal clades: planktonic diatoms and red macroalgae. The biosynthetic pathway to isodomoic acid A was recently solved in the harmful algal bloom-forming diatom Pseudonitzschia multiseries, establishing the genetic basis for the global production of this potent neurotoxin. Herein, we sequenced the 507-Mb genome of Chondria armata, the red macroalgal seaweed from which DA was first isolated in the 1950s, identifying several copies of the red algal DA (rad) biosynthetic gene cluster. The rad genes are organized similarly to the diatom DA biosynthesis cluster in terms of gene synteny, including a cytochrome P450 (CYP450) enzyme critical to DA production that is notably absent in red algae that produce the simpler kainoid neurochemical, kainic acid. The biochemical characterization of the N-prenyltransferase (RadA) and kainoid synthase (RadC) enzymes support a slightly altered DA biosynthetic model in C. armata via the congener isodomoic acid B, with RadC behaving more like the homologous diatom enzyme despite higher amino acid similarity to red algal kainic acid synthesis enzymes. A phylogenetic analysis of the rad genes suggests unique origins for the red macroalgal and diatom genes in their respective hosts, with native eukaryotic CYP450 neofunctionalization combining with the horizontal gene transfer of N-prenyltransferases and kainoid synthases to establish DA production within the algal lineages.

Identification and functional validation of super-enhancers in Arabidopsis thaliana.

Super-enhancers (SEs) are exceptionally large enhancers and are recognized to play prominent roles in cell identity in mammalian species. We surveyed the genomic regions containing large clusters of accessible chromatin regions (ACRs) marked by deoxyribonuclease (DNase) I hypersensitivity in Arabidopsis thaliana. We identified a set of 749 putative SEs, which have a minimum length of 1.5 kilobases and represent the top 2.5% of the largest ACR clusters. We demonstrate that the genomic regions associating with these SEs were more sensitive to DNase I than other nonpromoter ACRs. The SEs were preferentially associated with topologically associating domains. Furthermore, the SEs and their predicted cognate genes were frequently associated with organ development and tissue identity in A. thaliana. Therefore, the A. thaliana SEs and their cognate genes mirror the functional characteristics of those reported in mammalian species. We developed CRISPR/Cas-mediated deletion lines of a 3,578-bp SE associated with the thalianol biosynthetic gene cluster (BGC). Small deletions (131-157 bp) within the SE resulted in distinct phenotypic changes and transcriptional repression of all five thalianol genes. In addition, T-DNA insertions in the SE region resulted in transcriptional alteration of all five thalianol genes. Thus, this SE appears to play a central role in coordinating the operon-like expression pattern of the thalianol BGC.

A systematic comparison of natural product potential, with an emphasis on RiPPs, by mining of bacteria of three large ecosystems.

The implementation of several global microbiome studies has yielded extensive insights into the biosynthetic potential of natural microbial communities. However, studies on the distribution of several classes of ribosomally synthesized and post-translationally modified peptides (RiPPs), non-ribosomal peptides (NRPs) and polyketides (PKs) in different large microbial ecosystems have been very limited. Here, we collected a large set of metagenome-assembled bacterial genomes from marine, freshwater and terrestrial ecosystems to investigate the biosynthetic potential of these bacteria. We demonstrate the utility of public dataset collections for revealing the different secondary metabolite biosynthetic potentials among these different living environments. We show that there is a higher occurrence of RiPPs in terrestrial systems, while in marine systems, we found relatively more terpene-, NRP-, and PK encoding gene clusters. Among the many new biosynthetic gene clusters (BGCs) identified, we analyzed various Nif-11-like and nitrile hydratase leader peptide (NHLP) containing gene clusters that would merit further study, including promising products, such as mersacidin-, LAP- and proteusin analogs. This research highlights the significance of public datasets in elucidating the biosynthetic potential of microbes in different living environments and underscores the wide bioengineering opportunities within the RiPP family.

Plant terpene specialized metabolism: complex networks or simple linear pathways?

From the perspectives of pathway evolution, discovery and engineering of plant specialized metabolism, the nature of the biosynthetic routes represents a critical aspect. Classical models depict biosynthesis typically from an end-point angle and as linear, for example, connecting central and specialized metabolism. As the number of functionally elucidated routes increased, the enzymatic foundation of complex plant chemistries became increasingly well understood. The perception of linear pathway models has been severely challenged. With a focus on plant terpenoid specialized metabolism, we review here illustrative examples supporting that plants have evolved complex networks driving chemical diversification. The completion of several diterpene, sesquiterpene and monoterpene routes shows complex formation of scaffolds and their subsequent functionalization. These networks show that branch points, including multiple sub-routes, mean that metabolic grids are the rule rather than the exception. This concept presents significant implications for biotechnological production.

Unveiling biosynthetic potential of an Arctic marine-derived strain Aspergillus sydowii MNP-2.

BACKGROUND: A growing number of studies have demonstrated that the polar regions have the potential to be a significant repository of microbial resources and a potential source of active ingredients. Genome mining strategy plays a key role in the discovery of bioactive secondary metabolites (SMs) from microorganisms. This work highlighted deciphering the biosynthetic potential of an Arctic marine-derived strain Aspergillus sydowii MNP-2 by a combination of whole genome analysis and antiSMASH as well as feature-based molecular networking (MN) in the Global Natural Products Social Molecular Networking (GNPS). RESULTS: In this study, a high-quality whole genome sequence of an Arctic marine strain MNP-2, with a size of 34.9 Mb was successfully obtained. Its total number of genes predicted by BRAKER software was 13,218, and that of non-coding RNAs (rRNA, sRNA, snRNA, and tRNA) predicted by using INFERNAL software was 204. AntiSMASH results indicated that strain MNP-2 harbors 56 biosynthetic gene clusters (BGCs), including 18 NRPS/NRPS-like gene clusters, 10 PKS/PKS-like gene clusters, 8 terpene synthse gene clusters, 5 indole synthase gene clusters, 10 hybrid gene clusters, and 5 fungal-RiPP gene clusters. Metabolic analyses of strain MNP-2 grown on various media using GNPS networking revealed its great potential for the biosynthesis of bioactive SMs containing a variety of heterocyclic and bridge-ring structures. For example, compound G-8 exhibited a potent anti-HIV effect with an IC50 value of 7.2 nM and an EC50 value of 0.9 nM. Compound G-6 had excellent in vitro cytotoxicities against the K562, MCF-7, Hela, DU145, U1975, SGC-7901, A549, MOLT-4, and HL60 cell lines, with IC50 values ranging from 0.10 to 3.3 µM, and showed significant anti-viral (H1N1 and H3N2) activities with IC50 values of 15.9 and 30.0 µM, respectively. CONCLUSIONS: These findings definitely improve our knowledge about the molecular biology of genus A. sydowii and would effectively unveil the biosynthetic potential of strain MNP-2 using genomics and metabolomics techniques.

The porcine skin microbiome exhibits broad fungal antagonism.

The skin and its microbiome function to protect the host from pathogen colonization and environmental stressors. In this study, using the Wisconsin Miniature Swine™ model, we characterize the porcine skin fungal and bacterial microbiomes, identify bacterial isolates displaying antifungal activity, and use whole-genome sequencing to identify biosynthetic gene clusters encoding for secondary metabolites that may be responsible for the antagonistic effects on fungi. Through this comprehensive approach of paired microbiome sequencing with culturomics, we report the discovery of novel species of Corynebacterium and Rothia. Further, this study represents the first comprehensive evaluation of the porcine skin mycobiome and the evaluation of bacterial-fungal interactions on this surface. Several diverse bacterial isolates exhibit potent antifungal properties against opportunistic fungal pathogens in vitro. Genomic analysis of inhibitory species revealed a diverse repertoire of uncharacterized biosynthetic gene clusters suggesting a reservoir of novel chemical and biological diversity. Collectively, the porcine skin microbiome represents a potential unique source of novel antifungals.

Biosynthesis of Octacosamicin A: Uncommon Starter/extender Units and Product Releasing via Intermolecular Amidation.

Octacosamicin A is an antifungal metabolite featuring a linear polyene-polyol chain flanked by N-hydroxyguanidine and glycine moieties. We report here that sub-inhibitory concentrations of streptomycin elicited the production of octacosamicin A in Amycolatopsis azurea DSM 43854T . We identified the biosynthetic gene cluster (oca BGC) that encodes a modular polyketide synthase (PKS) system for assembling the polyene-polyol chain of octacosamicin A. Our analysis suggested that the N-hydroxyguanidine unit originates from a 4-guanidinobutyryl-CoA starter unit, while the PKS incorporates an α-hydroxyketone moiety using a (2R)-hydroxymalonyl-CoA extender unit. The modular PKS system contains a non-canonical terminal module that lacks thioesterase (TE) and acyl carrier protein (ACP) domains, indicating the biosynthesis is likely to employ an unconventional and cryptic off-loading mechanism that attaches glycine to the polyene-polyol chain via an intermolecular amidation reaction.

Biosynthesis of Arcyriaflavin F from Streptomyces venezuelae ATCC 10712.

Indolocarbazoles are natural products with broad bioactivities. A distinct feature of indolocarbazole biosynthesis is the modification of the indole and maleimide rings by regioselective tailoring enzymes. Here, we study a new indolocarbazole variant, which is encoded by the acfXODCP genes from Streptomyces venezuelae ATCC 10712. First we characterise this pathway by expressing the acfXODCP genes in Streptomyces coelicolor, which led to the production of a C-5/C-5'-dihydroxylated indolocarbazole. We assign as a new product arcyriaflavin F. Second, we demonstrate the flavin-dependent monooxygenase AcfX catalyses the C-5/C-5' dihydroxylation of the unsubstituted arcyriaflavin A into arcyriaflavin F. Interestingly, AcfX shares homology to EspX from erdasporine A biosynthesis, which instead catalyses a single C-6 indolocarbazole hydroxylation. In summary, we report a new indolocarbazole biosynthetic pathway and a regioselective C-5 indole ring tailoring enzyme AcfX.

Metabolic engineering of "last-line antibiotic" colistin in Paenibacillus polymyxa.

Colistin, also known as polymyxin E, is a lipopeptide antibiotic used to treat infections caused by multidrug-resistant gram-negative bacteria. It is considered a "last-line antibiotic", but its clinical development is hindered by low titer and impurities resulting from the presence of diverse homologs in microbial fermentation. To ensure consistent pharmaceutical activity and kinetics, it is crucial to have high-purity colistin active pharmaceutical ingredient (API) in the pharmaceutical industry. This study focused on the metabolic engineering of a natural colistin producer strain to produce colistin with a high titer and purity. Guided by genome mining, we identified Paenibacillus polymyxa ATCC 842 as a natural colistin producer capable of generating a high proportion of colistin A. By systematically inactivating seven non-essential biosynthetic gene clusters (BGCs) of peptide metabolites that might compete precursors with colistin or inhibit colistin production, we created an engineered strain, P14, which exhibited an 82% increase in colistin titer and effectively eliminated metabolite impurities such as tridecaptin, paenibacillin, and paenilan. Additionally, we engineered the L-2,4-diaminobutyric acid (L-2,4-DABA) pathway to further enhance colistin production, resulting in the engineered strain P19, which boosted a remarkable colistin titer of 649.3 mg/L - a 269% improvement compared to the original strain. By concurrently feeding L-isoleucine and L-leucine, we successfully produced high-purity colistin A, constituting 88% of the total colistin products. This study highlights the potential of metabolic engineering in improving the titer and purity of lipopeptide antibiotics in the non-model strain, making them more suitable for clinical use. These findings indicate that efficiently producing colistin API in high purity directly from fermentation can now be achieved in a straightforward manner.

Mining biosynthetic gene clusters in Paenibacillus genomes to discover novel antibiotics.

BACKGROUND: Bacterial antimicrobial resistance poses a severe threat to humanity, necessitating the urgent development of new antibiotics. Recent advances in genome sequencing offer new avenues for antibiotic discovery. Paenibacillus genomes encompass a considerable array of antibiotic biosynthetic gene clusters (BGCs), rendering these species as good candidates for genome-driven novel antibiotic exploration. Nevertheless, BGCs within Paenibacillus genomes have not been extensively studied. RESULTS: We conducted an analysis of 554 Paenibacillus genome sequences, sourced from the National Center for Biotechnology Information database, with a focused investigation involving 89 of these genomes via antiSMASH. Our analysis unearthed a total of 848 BGCs, of which 716 (84.4%) were classified as unknown. From the initial pool of 554 Paenibacillus strains, we selected 26 available in culture collections for an in-depth evaluation. Genomic scrutiny of these selected strains unveiled 255 BGCs, encoding non-ribosomal peptide synthetases, polyketide synthases, and bacteriocins, with 221 (86.7%) classified as unknown. Among these strains, 20 exhibited antimicrobial activity against the gram-positive bacterium Micrococcus luteus, yet only six strains displayed activity against the gram-negative bacterium Escherichia coli. We proceeded to focus on Paenibacillus brasilensis, which featured five new BGCs for further investigation. To facilitate detailed characterization, we constructed a mutant in which a single BGC encoding a novel antibiotic was activated while simultaneously inactivating multiple BGCs using a cytosine base editor (CBE). The novel antibiotic was found to be localized to the cell wall and demonstrated activity against both gram-positive bacteria and fungi. The chemical structure of the new antibiotic was elucidated on the basis of ESIMS, 1D and 2D NMR spectroscopic data. The novel compound, with a molecular weight of 926, was named bracidin. CONCLUSIONS: This study outcome highlights the potential of Paenibacillus species as valuable sources for novel antibiotics. In addition, CBE-mediated dereplication of antibiotics proved to be a rapid and efficient method for characterizing novel antibiotics from Paenibacillus species, suggesting that it will greatly accelerate the genome-based development of new antibiotics.

Amycolatopsis mongoliensis sp. nov., a novel actinobacterium with antifungal activity isolated from a coal mining site in Mongolia.

A novel filamentous actinobacterium designated strain 4-36T showing broad-spectrum antifungal activity was isolated from a coal mining site in Mongolia, and its taxonomic position was determined using polyphasic approach. Optimum growth occurred at 30 °C, pH 7.5 and in the absence of NaCl. Aerial and substrate mycelia were abundantly formed on agar media. The colour of aerial mycelium was white and diffusible pigments were not formed. Phylogenetic analyses based on 16S rRNA gene sequence showed that strain 4-36T formed a distinct clade within the genus Amycolatopsis. The 16S rRNA gene sequence similarity showed that the strain was mostly related to Amycolatopsis lexingtonensis DSM 44544T and Amycolatopsis rifamycinica DSM 46095T with 99.3 % sequence similarity. However, the highest digital DNA-DNA hybridization value to closest species was 44.1 %, and the highest average nucleotide identity value was 90.2 %, both of which were well below the species delineation thresholds. Chemotaxonomic properties were typical of the genus Amycolatopsis, as the major fatty acids were C15 : 0, iso-C16 : 0 and C16 : 0, the cell-wall diamino acid was meso-diaminopimelic acid, the quinone was MK-9(H4), and the main polar lipids were diphosphatidylglycerol, phosphatidylmethanolamine and phosphatidylethanolamine. The in silico prediction of chemotaxonomic markers was also carried out by phylogenetic analysis. The genome mining for biosynthetic gene clusters of secondary metabolites in strain 4-36T revealed the presence of 34 gene clusters involved in the production of polyketide synthase, nonribosomal peptide synthetase, ribosomally synthesized and post-translationally modified peptide, lanthipeptide, terpenes, siderophore and many other unknown clusters. Strain 4-36T showed broad antifungal activity against several filamentous fungi. The phenotypic, biochemical and chemotaxonomic properties indicated that the strain could be clearly distinguished from other species of Amycolatopsis, and thus the name Amycolatopsis mongoliensis sp. nov. is proposed accordingly (type strain, 4-36T=KCTC 39526T=JCM 30565T).

Non-ribosomal peptide synthetase (NRPS)-encoding products and their biosynthetic logics in Fusarium.

Fungal non-ribosomal peptide synthetase (NRPS)-encoding products play a paramount role in new drug discovery. Fusarium, one of the most common filamentous fungi, is well-known for its biosynthetic potential of NRPS-type compounds with diverse structural motifs and various biological properties. With the continuous improvement and extensive application of bioinformatic tools (e.g., anti-SMASH, NCBI, UniProt), more and more biosynthetic gene clusters (BGCs) of secondary metabolites (SMs) have been identified in Fusarium strains. However, the biosynthetic logics of these SMs have not yet been well investigated till now. With the aim to increase our knowledge of the biosynthetic logics of NPRS-encoding products in Fusarium, this review firstly provides an overview of research advances in elucidating their biosynthetic pathways.

Genomic characterization of three bacterial isolates antagonistic to the pea root rot pathogen Aphanomyces euteiches.

Microorganisms living in soil and rhizosphere or inside plants can promote plant growth and health. Genomic characterization of beneficial microbes could shed light on their special features. Through extensive field survey across Saskatchewan, Canada, followed by in vitro and greenhouse characterization, we identified several bacterial isolates antagonistic to pea root rot pathogen Aphanomyces euteiches. In this study, the genomes of three isolates-Pseudomonas sp. rhizo 66 (PD-S66), Pseudomonas synxantha rhizo 25 (Ps-S25), and Serratia sp. root 2 (TS-R2)-were sequenced, assembled, and annotated. Genome size of PD-S66 was 6 279 416 bp with 65 contigs, 59.32% GC content, and 5653 predicted coding sequences (CDS). Genome size of Ps-S25 was 6 058 437 bp with 66 contigs, a GC content of 60.08%, and 5575 predicted CDS. The genome size of TS-R2 was 5 282 152 bp, containing 26 contigs, a GC content of 56.17%, and 4956 predicted CDS. For the identification of the isolates, digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values were determined, which confirmed PD-S66 and TS-R2 as potential new species, belonging to Pseudomonas and Serratia genera, respectively, while Ps-S25 belongs to species Pseudomonas synxantha. Biosynthetic gene clusters were predicted using antiSMASH. The genomic data provided insight into the genetics and biochemical pathways supporting the antagonistic activity against A. euteiches of these isolates.

CRISPR-aided genome engineering for secondary metabolite biosynthesis in Streptomyces.

The demand for discovering novel microbial secondary metabolites is growing to address the limitations in bioactivities such as antibacterial, antifungal, anticancer, anthelmintic, and immunosuppressive functions. Among microbes, the genus Streptomyces holds particular significance for secondary metabolite discovery. Each Streptomyces species typically encodes approximately 30 secondary metabolite biosynthetic gene clusters (smBGCs) within its genome, which are mostly uncharacterized in terms of their products and bioactivities. The development of next-generation sequencing has enabled the identification of a large number of potent smBGCs for novel secondary metabolites that are imbalanced in number compared with discovered secondary metabolites. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has revolutionized the translation of enormous genomic potential into the discovery of secondary metabolites as the most efficient genetic engineering tool for Streptomyces. In this review, the current status of CRISPR/Cas applications in Streptomyces is summarized, with particular focus on the identification of secondary metabolite biosynthesis gene clusters and their potential applications.This review summarizes the broad range of CRISPR/Cas applications in Streptomyces for natural product discovery and production. ONE-SENTENCE SUMMARY: This review summarizes the broad range of CRISPR/Cas applications in Streptomyces for natural product discovery and production.

Discovery of Streptomyces species CS-62, a novel producer of the Acinetobacter baumannii selective antibiotic factumycin.

Narrow-spectrum antibiotics are of great interest given their ability to spare the microbiome and decrease widespread antibiotic resistance compared to broad-spectrum antibiotics. Herein, we screened an in-house library of Actinobacteria strains for selective activity against Acinetobacter baumannii and successfully identified Streptomyces sp. CS-62 as a producer of a natural product with this valuable activity. Analysis of the cultures via high-resolution mass spectrometry and tandem mass spectrometry, followed by comparison with molecules in the Natural Product Atlas and the Global Natural Products Social Molecular Networking platform, suggested a novel natural product. Genome mining analysis initially supported the production of a novel kirromycin derivative. Isolation and structure elucidation via mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses revealed that the active natural product was the known natural product factumycin, exposing omissions and errors in the consulted databases. While public databases are generally very useful for avoiding rediscovery of known molecules, rediscovery remains a problem due to public databases either being incomplete or having errors that result in failed dereplication. Overall, the work describes the ongoing problem of dereplication and the continued need for public database curation.

Genomic Analysis of Novel Sulfitobacter Bacterial Strains Isolated from Marine Biofilms.

Bacteria from the genus Sulfitobacter are distributed across various marine habitats and play a significant role in sulfur cycling. However, the metabolic features of Sulfitobacter inhabiting marine biofilms are still not well understood. Here, complete genomes and paired metatranscriptomes of eight Sulfitobacter strains, isolated from biofilms on subtidal stones, have been analyzed to explore their central energy metabolism and potential of secondary metabolite biosynthesis. Based on average nucleotide identity and phylogenetic analysis, the eight strains were classified into six novel species and two novel strains. The reconstruction of the metabolic pathways indicated that all strains had a complete Entner-Doudoroff pathway, pentose phosphate pathway, and diverse pathways for amino acid metabolism, suggesting the presence of an optimized central carbon metabolism. Pangenome analysis further revealed the differences between the gene cluster distribution patterns among the eight strains, suggesting significant functional variation. Moreover, a total of 47 biosynthetic gene clusters were discovered, which were further classified into 37 gene cluster families that showed low similarity with previously documented clusters. Furthermore, metatranscriptomic analysis revealed the expressions of key functional genes involved in the biosynthesis of ribosomal peptides in in situ marine biofilms. Overall, this study sheds new light on the metabolic features, adaptive strategies, and value of genome mining in this group of biofilm-associated Sulfitobacter bacteria.

Comparative pangenome analysis of Aspergillus flavus and Aspergillus oryzae reveals their phylogenetic, genomic, and metabolic homogeneity.

Aspergillus flavus and Aspergillus oryzae are closely related fungal species with contrasting roles in food safety and fermentation. To comprehensively investigate their phylogenetic, genomic, and metabolic characteristics, we conducted an extensive comparative pangenome analysis using complete, dereplicated genome sets for both species. Phylogenetic analyses, employing both the entirety of the identified single-copy orthologous genes and six housekeeping genes commonly used for fungal classification, did not reveal clear differentiation between A. flavus and A. oryzae genomes. Upon analyzing the aflatoxin biosynthesis gene clusters within the genomes, we observed that non-aflatoxin-producing strains were dispersed throughout the phylogenetic tree, encompassing both A. flavus and A. oryzae strains. This suggests that aflatoxin production is not a distinguishing trait between the two species. Furthermore, A. oryzae and A. flavus strains displayed remarkably similar genomic attributes, including genome sizes, gene contents, and G + C contents, as well as metabolic features and pathways. The profiles of CAZyme genes and secondary metabolite biosynthesis gene clusters within the genomes of both species further highlight their similarity. Collectively, these findings challenge the conventional differentiation of A. flavus and A. oryzae as distinct species and highlight their phylogenetic, genomic, and metabolic homogeneity, potentially indicating that they may indeed belong to the same species.

Isolation and Structure Elucidation of JBIR-157, a Skeletally Novel Aromatic Polyketide Produced by the Heterologous Expression of a Cryptic Gene Cluster.

Heterologous expression of natural compound biosynthetic gene clusters (BGCs) is a robust approach for not only revealing the biosynthetic mechanisms leading to the compounds, but also for discovering new products from uncharacterized BGCs. We established a heterologous expression technique applicable to huge biosynthetic gene clusters for generating large molecular secondary metabolites such as type-I polyketides. As an example, we targeted concanamycin BGC from Streptomyces neyagawaensis IFO13477 (the cluster size of 99 kbp), and obtained a bacterial artificial chromosome (BAC) clone with an insert size of 211 kbp that contains the entire concanamycin BGC. Interestingly, heterologous expression for this BAC clone resulted in two additional aromatic polyketides, ent-gephyromycin, and a new compound designated as JBIR-157, together with the expected concanamycin. Bioinformatic and biochemical analyses revealed that a cryptic biosynthetic gene cluster in this BAC clone was responsible for the production of these type-II polyketide synthases (PKS) compounds. Here, we describe the production, isolation, and structure elucidation of JBIR-157, determined primarily by a series of NMR spectral analyses.